This project is designed to elucidate the role of the serine-threonine kinases protein kinase CK2 (Casein kinase II) and glycogen synthase kinase-3 (GSK3) in mammary development and tumorigenesis pathways. These were among the first kinases identified by Edwin Krebs and colleagues in the 1950?s, but we still do not fully understand their roles in the control of cell growth and differentiation. Although CK2 is capable of phosphorylating casein, this is not its physiological function. In fact, CK2 and GSK3 are both involved in the coordinated phosphorylation of glycogen synthase, and thus, regulate a critical step of intermediary metabolism, and both phosphorylate and regulate a variety of transcription factors. Of particular relevance to mammary tumorigenesis, GSK3 is a negative regulator of the "Wnt" growth factor signaling pathway. Recently, the Project Leader?s laboratory has recently demonstrated that CK2 is a positive regulator of Wnt signaling through phosphorylation and stabilization of beta-catenin, the essential co-factor for TCF/LEF dependent transcription in response to Wnt signals. Wnt, beta-catenin, and their target genes play an important role in developmental pathways, in mouse mammary tumor virus (MMTV) induced breast cancer in mice, and in a variety of cancers in humans, including breast cancer. We hypothesize that CK2 and GSK3 play roles in normal mammary development and in the molecular pathogenesis of breast cancer, and we will test this hypothesis in in vitro and in vivo systems. The Specific Aims of this project are to: (1) characterize CK2 and GSK3 expression and activity and wnt pathway activation during normal mouse mammary gland development and in breast cancer using specimens of human breast tumors and carcinogen-induced rat tumors; (2) determine the consequences of manipulation of CK2 and GSK3 levels on wnt signaling and growth of mammary cell lines in vitro; and (3) carry out similar studies in vivo, using transgenic mice that we have engineered that overexpress either CK2 or GSK3 in the mammary gland and develop breast Cancer. Through this program project, access will be available to: (a) specimens from human breast tumors to validate the results in the mouse models; (b) mammary gland specimens from rats treated with carcinogens, allowing longitudinal study of expression of these genes in the process of tumorigenesis; and reagents and expertise in pathways with which these enzymes may interact. Preliminary results implicate the NF-kB pathway, the focus of Project 3, as one important target of CK2 activity in mammary epithelial cells. The long-term goal is that an understanding of the role of these regulatory kinases in the molecular pathogenesis of breast cancer may eventually lead to novel anti-cancer therapies.